In a normal blood, erythrocytes are about one thousand times more abundant than leucocytes and form an obstacle to any study or analysis of leucocytes. This is why methods of leucocyte analysis such as cytometry, or cytospin involving centrifugation of the blood constituents, are preceded in most cases by a step for separating erythrocytes from leucocytes (U.S. Pat. No. 4,284,412, EPA-0 022 670).
One such separation by centrifugation using Ficoll to isolate the lymphocytes and granulocytes from the blood has been described by Boyem in Scandinavian Journal of Clinical and Laboratory Investigation 1967-68, Suppl. 94-101.305.293 and Pertoff in Journal of Immunological Methods: 1980, 33:221-229.
These methods are relatively difficult to use because they require several washes and centrifugations, which may lead to losses of cells and hence to an assay by default. However, said methods are currently considered to be reference methods since the morphology and viability of the leucocytes are preserved after separation.
The lysis of blood with a view to a leucocyte analysis involves the destruction of the red corpuscles to a state of debris. The lysis methods, on the other hand, aim for optimum preservation of the morphology of the leucocytes in order to permit a cytometric leucocyte analysis and to distinguish the leucocytes clearly from the erythrocyte debris.
The lysis of erythrocytes is based on the general principle of passing a quantity of material through the membrane from the outside of the erythrocyte. During this passage, the condition of the membrane deteriorates either due to swelling of the cell or due to dissolution of the membrane in the material passing through.
The preservation of the leucocyte morphology during lysis is based on a more protein-rich environment around its membrane: outside and inside, transmembrane proteins, and inside, a developed cytoskeleton.
The advantage of this leucocyte membrane support is exploited to the full by the use of fixing agents such as formaldehyde which penetrates the membrane and fixes the protein structures by crosslinking outside and inside.
The material used for passing through the erythrocyte membranes are generally small neutral molecules, firstly water itself, used in hypotonic lysis, or water in the presence of diethylene glycol, formaldehyde and citric acid as in Chang et al. (U.S. Pat. No. 4,902,613). Sometimes, fixing and hypotonic lysis are carried out at different stages as described by Quintana (WO-89/0509) and van Agthoven (EPA-0625706).
In the case of isotonic lyses as described in EP-A-625 707, the material passed through is composed of a mixture of formaldehyde, glycerol, butanol and citric acid.
Another method of isotonic lysis uses saponin, a small molecule with detergent properties as described in WO 85/05640.
There is a method of lysis wherein the general principle of lysis is not immediately apparent. This is lysis with ammonium chloride. The lysis operation depends on the passage of NH.sub.3 and CO.sub.2 through the membrane. NH.sub.3 and CO.sub.2 are in equilibrium with NH.sub.4.sup.+ and HCO.sub.3.sup.- in the mixture. The spontaneous re-conversion of NH.sub.3 to NH.sub.4.sup.+ in the cell and of CO.sub.2 to HCO.sub.3.sup.- by carbonic anhydrase present in large quantities in the erythrocyte could be the force behind a continuous flow of NH.sub.3 and CO.sub.2 entering the cell.
Lysis with ammonium chloride is the most effective lysis that can be carried out in the absence of fixing agent and it is therefore the preferred method of many research laboratories. A limitation of this method is that the leucocytes very quickly lose their viability during the lysis process. Apparently, the interior of the cell is initially rendered alkaline by the product of the two reactions; NH.sub.5 CO.sub.3. Later on, in the reaction after lysis and release of carbonic anhydrase from the erythrocytes, the reaction HCO.sub.3 .fwdarw.CO.sub.2 +OH.sup.- from the bicarbonate present in the lysis buffer renders the entire mixture alkaline, which brings about the degradation of the leucocytes. Due to these toxic conditions, the reading must be taken very rapidly, usually 1 to 2 hours after carrying out the preparation.
Another limitation of the method is the fact that it is impossible to combine the use of ammonium chloride with formaldehyde due to the following reaction: EQU 6HCHO+4NH.sub.3 .fwdarw.C.sub.6 H.sub.12 N.sub.4 +6H.sub.2 O.
The hexamethylene tetramine formed is stable and consequently the mixture acidifies during the reaction to the detriment of the leucocytes because the removal of NH.sub.3 in the medium shifts the equilibrium and the H.sup.+ 0 ions are no longer compensated for.
To summarise, ammonium chloride has proved to be incompatible with aliphatic aldehydes and consequently lysis stops rapidly when both are used together.